The polymerase chain reaction has, since its conception in April 1983, become a standard technique for amplification of nucleic acid sequences. In this process one or more specific nucleic acid sequences present in a nucleic acid or mixture thereof are amplified using primers and agents for polymerization and then the amplified sequence is detected. The extension product of one primer, when hybridized to the other, becomes a template for the production of the desired specific nucleic acid sequence, and vice versa, and the process is repeated as often as is necessary to produce the desired amount of the sequence.
The DNA or RNA may be single- or double-stranded, and may be a relatively pure species or a component of a mixture of nucleic acids. The PCR process utilizes a repetitive reaction to accomplish the amplification of the desired nucleic acid sequence. Indeed, in the approximately eleven years since its discovery, the PCR technique has been widely employed in the field of molecular biology in detecting and identifying nucleic acid sequences. For example, the polymerase chain reaction (PCR) has been extensively used to amplify DNA loci in genome mapping, linkage studies, genetic diagnostics, forensics, and paternity testing. The wide-spread use of this technique is testimony to the utility of the process.
Unfortunately, the polymerase chain reaction is not without problems. The PCR process depends upon multiple steps of melting and annealing (separation of two complementary strands). Under ideal conditions, one can envision the separation of the two complementary strands of double-stranded DNA, annealing of the complementary primers, and elongation of the primers with a polymerase to produce two molecules of double-stranded DNA. This cycle is repeated while the quantity of the desired sequence grows exponentially.
However, in most contexts in which the polymerase chain reaction is employed, situations are less than ideal. Often DNA of interest to the biotechnological community contains a number of repeat or near-repeat sequences. Additionally, nucleic acid sequences frequently contain a number of internally self-complementary sequences. These repeating and self-complementary sequences increase the likelihood that the nucleic acid sequence will experience interstrand and/or intrastrand interactions, including the possibility of a nucleic acid strand folding upon itself. As fragments of interest often tend to be on the order of about 600 base pairs or longer in length and often about 10-40 Kb (or more) in length, the likelihood of such interactions is increased.
Further, as the polymerase chain reaction progresses, the concentration of complementary DNA sequences increases exponentially, while the concentration of primer commonly remains constant or decreases. Thus, as the reaction progresses the ratio of primer to template decreases. This can result in the product of the interim cycles of the polymerase chain reaction successfully competing for the template required for the next cycle of elongation. Such competition can significantly reduce the efficiency and yield of the polymerase chain reaction and may result in the generation of anomalous product and limit the length of DNA successfully traversed by the polymerase.
Moreover, diploid DNA poses additional challenges. Robust amplification of allelic sequences is often difficult, particularly when there is a significant size difference between the two alleles which are present in the heterozygous state. Smaller alleles simply amplify more efficiently. This phenomenon, known as differential or preferential amplification, results in the generation of more copies of the smaller allele and thus a relatively more intense band on the subsequent electrophoresis gel. At best, the larger allele is underrepresented relative to the smaller allele, at worst, differential amplification can result in allelic dropout, in which the larger allele amplifies so poorly relative to the smaller allele that the larger allele can neither be visualized nor detected. Differential amplification and allelic dropout can complicate genetic analyses.
Preferential amplification limits the usefulness of the PCR when amplifying VNTR loci. No truly satisfactory mechanisms have been proposed to explain the differential, amplification phenomenon in PCR. Walsh, et al., in PCR Methods and Applications, p.241 (1992) suggested that (in some cases) preferential amplification may be the result of incomplete denaturation, differential priming, limiting enzyme or small sample size.
As alluded to above, other problems associated with PCR include the generation of anomalous product (or ladder-like patterns) and template length limitations. Mechanisms proposed for anomalous product generation include slipped strand mispairing (Levinson, G. and Gutman, G. A. (1987) Molec. Biol. Evol., 4, 203-221; Tautz, D. (1989) Nucl. Acids Res., 17, 6463-6471; and Litt, M. and Luty, J. A. (1989) Am. J. Hum. Genet., 44, 397-401), the addition of nucleotides to the 3' end of the template (Hu, G. (1993) DNA Cell Biol., 12 763-770) and truncated PCR product (Meyerhans, A., Vartanian, J. P. and Wain-Hobson, S. (1990) Nucl. Acids Res., 18, 1687-1691, and Marton, A., Delbecchi, L. and Bourgaux, P. (1991) Nucl. Acids Res., 19, 2423-2426), or "out of register" template switching (Odelberg, S., Weiss, R. B., Hata, A., and White, R. (1991) Crime Lab. Digest, 18:4, 137, Odelberg S. and White R. (1993) PCR Meth. Appl., 3, 7-12, and Odelberg, S., Weiss, R. B., Hata, A., and White, R. supra). Mechanisms proposed to explain the length limitations experienced in PCR include premature termination of primer extension due to template nicking, incorporation of mismatched base pairs, and premature dissociation of polymerase (Barnes, W. M. (1994) Proc. Natl. Acad. Sci. 91, 2216-2220; Cheng, S., Fockler, C., Barnes, W. M. and Higuchi, R. (1994) Proc. Natl. Acad. Sci. 91, 5695-5699; Foord, O. S. and Rose, E. A. (1994) PCR Meth. Appl. 3, S149-S161).
These limitations of PCR--preferential amplification, anomalous product generation and template length limitations--have each been considered in isolation by various investigators, but never conjunctively and never with any recognition of any interrelationship between them.
Recently, methodology for improving the PCR by reducing the length limitations of the templates which can be used has been developed. These include buffer modification (such as pH) and reduction of template nicking (Cheng, S, Fockler, C, Barnes, W. M. and Higuchi, R. (1994) Proc. Natl. Acad. Sci. 91, 5695-5699) and premature termination of primer extension due to misincorporation of mismatched base pairs (Barnes, W. M. (1994) Proc. Natl. Acad. Sci. 91, 2216-2220) Use of these methodologies separately or conjunctively has been shown to facilitate the PCR amplication of long templates. Such methodology has come to be referred to by artisans as "long PCR methodology."
The terms "peptide nucleic acid" and "PNA" refer to a DNA analog with a backbone consisting of N-(2-aminoethyl)glycine units. To this backbone, analogous to DNA, are attached the nucleobases--for DNA, adenine, guanine, cytidine, and thymine. The individual monomeric units of PNA can be synthesized to furnish a PNA chain having a specific sequence of bases. The synthesis of such PNA chains is detailed in various publications, including Science 254, 1497 (1991); J. Am. Chem. Soc. 114, 9677 (1992); J. Am. Chem. Soc. 144, 1895 (1992); J. Chem. Soc. Chem. Comm. 800 (1993); Proc. Nat. Acad. Sci. USA 90, 1667 (1993); Intercept Ltd. 325 (1992); J. Am. Chem. Soc. 114, 9677 (1992); Nucleic Acids Res. 21, 197 (1993); J. Chem. Soc. Chem. Commun. 518 (1993); Anti-Cancer Drug Design 8, 53 (1993); Nucleic Acids Res. 21, 2103 (1993); Org. Proc. Prep. 25, 457 (1993); CRC Press 363 (1992); J. Chem. Soc. Chem. Commun. 9:800 (1993); J. Am. Chem. Soc. 115, 6477 (1993); Nature 365, 566 (1993); ABRF News Vol. 4, No. 3 (1993); Science 258, 1481 (1992); WO 8-92/20702; and WO 92/20703, the contents of which are incorporated herein by reference. Additionally, specific sequences of PNA are commercially available from BioSearch Div., PerSeptive Biosystems, Inc., Farmingham, Mass.
PNA has been demonstrated to be a potent DNA mimic in terms of sequence-specific annealing. Experimental results (discussed in the above publications) have demonstrated that at physiological ionic strength a PNA/DNA duplex is generally 1.degree. C. per base pair more stable thermally than the corresponding DNA/DNA duplex. Other experimental data has indicated that PNA is more stable in the cell than DNA; that PNA binds to DNA or RNA 50-100 times more tightly than either DNA or RNA; that PNA can invade and displace double-stranded DNA (dsDNA); and that the backbone of PNA adopts a helical conformation. However, a single base mismatch in a PNA/DNA duplex is much more destabilizing than a mismatch in the corresponding DNA/DNA duplex. Furthermore, PNA does not function as a primer for DNA polymerase.
Recently, PNAs have been used to detect single base mutations through PCR clamping. In Nucleic Acids Research 21, 5332 (1993), PNAs were reported to form a PNA/DNA complex which effectively blocked the formation of a PCR product. However, this PNA-directed PCR clamping results in a blocking of PCR amplification, rather than an enhancement of the polymerase chain reaction.
Accordingly, there remains a need for an efficient and effective method of amplifying existing nucleic acid sequences present in a sample using the polymerase chain reaction and for a method whereby PCR may proceed with enhanced efficiency whereby larger templates, and repeat sequences such as STRs and LTRs may be amplified while limiting preferential amplification or anomalous product generation.